1. Field of the Invention
The present invention relates to the field of biologically active peptide containing compositions for use in the prevention and treatment of hematopoietic neoplastic diseases, particularly leukemia.
2. Description of Related Art
LFA-1 (lymphocyte function associated antigen-1) is an integrin xcex1xcex2 heterodimer (Carlos and Harlan, 1994; Springer, 1994; Larson and Springer, 1990; McEver, 1990; Picker and Butcher, 1992). Although three other integrins restricted in expression to leukocytes share the same xcex2 subunit and have homologous xcex1 subunits (Mac-1, p150,95, and alpha d), only LFA-1 is expressed on normal and leukemia T cells (Larson and Springer, 1990). LFA-1 binds ICAM-1 (intracellular adhesion molecule), and although LFA-1 is constitutively expressed on all leukocytes, LFA-1 binding to ICAM-1 requires cellular activation. Activation, in part, results in conformational changes in LFA-1 that affect its avidity for ICAM-1. In contrast, ICAM-1 is constitutively avid and expressed on a wide array of cell types including leukocytes, endothelium, stromal cells, and fibroblasts. In a model developed by the present inventor, a stromal cell derived soluble factor cooperates with LFA-1 on the surface of T lineage acute lymphoblastic leukemia (T-ALL) cells (Winter et al., 1998). The LFA-1 on T-ALL cells results in bone marrow (BM) stromal cell binding via ICAM-1 that leads to enhanced leukemia cell survival. Furthermore, aberrant LFA-1/ICAM-1 dependent interaction between circulating leukemia cells and endothelial cells lining blood vessels promotes extravasation of leukemia cells into tissue as seen in the life-threatening therapeutic complication of acute leukemia, retinoic acid syndrome (Brown et al., 1999). Hence, the development of effective in vivo inhibitors of LFA-1/ICAM interaction would be useful in the therapy of acute leukemia and prevention of therapeutic complications.
The present inventor has shown, for example, that inhibition of LFA-1/ICAM-1 dependent stromal cell binding with mAbs decreases survival of T-ALL cell lines and T-ALL cells isolated from patients. In one study, a representative sample from a patient with T-ALL showed that survival of T-ALL cells is augmented by BM stromal cells and that survival is inhibited by mAbs directed against LFA-1 (mAb TSI/22,5 xcexcg/ml) or its ligand ICAM-1 (mAb 84H10, 10 xcexcg/ml). This observation has been replicated for T-ALL cell lines Jurkat and Sup T I as well as a subset of patients with T-ALL. However, even though in vivo use of mAbs against LFA-1 or ICAM-1 blocks LFA-1 function in a number of disease models, unfortunately anaphylactic reactions and secondary physiologic effects have hampered this approach (McMuray, 1996; DeMeester: et al., 1996; Jackson et al., 1997; Cuthbertson et al., 1997; Gundel et al., 1992; Haming et al., 1993; Nakano et al, 1995).
Another means to interfere with proteinxe2x80x94protein interactions is through the use of small peptide inhibitors. In fact, small peptide inhibitors to adhesion molecules structurally-related to LFA-1 have recently been approved for clinical use in coagulopathies (Ohman et al., 1995; Adgey et al., 1998; Leficovis and Topol, 1995). Short linear peptides ( less than 30 amino acids) have also been described that prevent or interfere with integrin dependent firm adhesion using sequences derived from integrin or their ligands. In particular, these peptides have been derived from a number of integrin receptors: the xcex22 and xcex23 subunits of integrins, and the xcex1iib subunit of ICAM-1, and VCAM-1 (Murayama et al., 1996; Jacobsson and Frykberg, 1996; Zhang and Plow, 1996; Budnik et al., 1996; Vanderslice et al, 1997; Suehiro et al., 1996; Endemann et al., 1996). However, the clinical applicability of these linear peptides is limited. The half maximal inhibitory concentration (IC50; concentration at which aggregation is inhibited 50%) for most of these peptides is 10xe2x88x924 M with purified receptor-ligand pairs (univalent interactions) and they are ineffective at inhibiting multivalent interactions, during cellxe2x80x94cell adhesion. In addition, linear peptides have short serum half-lives because of proteolysis. Therefore, prohibitively high concentrations of peptide would have to be administered in a clinical setting and a biologic effect would not necessarily occur.
Longer peptides, ranging in length from 25-200 residues, have also been reported to block xcex21, xcex22, and xcex23 integrin dependent adhesion (Zhang and Plow, 1996; Budnik et al., 1996; Vanderslice et al, 1997; Suehiro et al., 1996; Endeman et al., 1996). In general, these peptide inhibitors may have higher affinities or slower off-rates than short peptides and, therefore, are better inhibitors. However, they are still susceptible to proteolysis.
Therefore, a need exists to develop novel and specific classes of pharmaceutical agents to inhibit the binding of LFA-1 and ICAM-1 and to be useful in the treatment of hematopoietic neoplastic diseases as well as other diseases that involve emigration of leukocytes from blood into tissue, such as myocardial infarction, radiation injury, asthma, rheumatoid arthritis, and lymphoma metastasis.
The present invention addresses the problems in the art by providing compositions that include cyclic peptide inhibitors of binding interactions between the integrin, lymphocyte function associated antigen-1 (LFA-1) expressed on leukocytes, including leukemic T-cells, and intracellular adhesion molecule 1 (ICAM-1), expressed on a variety of cell types. As stated above, this binding of activated LFA-1 is implicated in a variety of diseases and inhibition of this binding interaction with a cyclic peptide inhibitor will have implications in the treatment or management of those diseases.
As a part of the present invention, phage display has been used to identify peptide sequences that bind ICAM-1 and block LFA-1/ICAM interaction. Phage that specifically bound ICAM-1 were identified by repeated selection from a cysteine-constrained heptapeptide phage display library. The peptide sequences expressed on ICAM-1 binding phage were determined by nucleotide sequencing. A consensus sequence, CLLRMRSIC (SEQ ID NO:1) was derived from the analysis of the most frequently occurring sequences. Analysis of less frequently recurring amino acids of ICAM-1 binding-phage identified variants of SEQ ID NO: 1 wherein the second amino acid is methionine (SEQ ID NO: 17), or in which the 5th amino acid is proline (SEQ ID NO: 5), or in which the 6th amino acid is asparagine (SEQ ID NO: 9), or in which the 7th amino acid is leucine (SEQ ID NO: 3), or in which the 8th amino acid is arginine (SEQ ID NO: 2), or any combination of these substitutions.
Another aspect of the present disclosure is the application of an alanine screening technique to the consensus sequence, SEQ ID NO: 1. An alanine was substituted at each position in the hepatapeptide LLRMRSI of the consensus sequence SEQ ID NO: 1, and each peptide was examined in for its ability to inhibit LFA-1/ICAM-1 mediated cell aggregation. Alanine substitution of the isoleucine at position 8 of SEQ ID NO: 1, i.e., CLLRMRSAC (SEQ ID NO: 40), resulted in a more potent antagonist. Loss of inhibitory function resulted from alanine substitution of the leucines in positions 2 (SEQ ID NO: 34) and 3 (SEQ ID NO: 35), methionine in position 5 (SEQ ID NO: 37), and arginine in position 6 (SEQ ID NO: 38) of SEQ ID NO: 1, indicating that these amino acids are important to the antagonistic activity of the peptide. Substitution of the serine in position 7 (SEQ ID NO: 39) had no significant effect on the inhibitory activity of the peptide. Substitution of the arginine in position 4 (i.e., SEQ ID NO: 36) of SEQ ID NO: 1 was not soluble in aqueous solution at 1 nm and was not tested.
A further aspect of the present invention is the use of conservative amino acid substitutions of the key amino acid residues identified by the alanine screen. Sequences that exhibit greater inhibition of LFA-1/ICAM-1 mediated cell inhibition as compared to SEQ ID NO: 1, include CILRMRSAC (SEQ ID NO: 43), CLIRMRSAC (SEQ ID NO: 44), CLLKMRSAC (SEQ ID NO 45), CLLRMKSAC (SEQ ID NO: 46), and CLLRMRSVC (SEQ ID NO: 48).
The present invention may be described, therefore, in certain aspects as a composition comprising a cyclic peptide inhibitor of LFA-1/ICAM-1 interaction, wherein the composition has the amino acid sequence, CLLRMRSIC (SEQ ID NO: 1) or CLLRMRSAC (SEQ ID NO: 40), or a conservative variant thereof. Conservative variants are described elsewhere herein, and include the exchange of an amino acid for another of like charge, size, or hydrophobicity, and include, but are not limited to, CILRMRSAC (SEQ ID NO: 43), CLIRMRSAC (SED ID NO: 44), CLLKMRSAC (SEQ ID NO 45), CLLRMKSAC (SEQ ID NO: 46), and CLLRMRSVC (SEQ ID NO: 48). The present disclosure would also include variants of SEQ ID NO: 1 in which the 3rd and 4th amino acids remain the same and other amino acids of the sequence are substituted and tested empirically for their ability to inhibit the LFA-1/ICAM-1 interaction. The amino acid sequence is numbered in the conventional sense, in that the first amino acid on the N-terminus is cysteine, followed by 7 amino acids and then a carboxy terminal cysteine. In the practice of the invention, the two terminal cysteines may form a disulfide bonded cystine residue resulting in a cyclic peptide as is well known in the art. Alternatively, the terminal cysteines may be linked by an amide peptide linkage, either directly or separated by one or more amino acids, leaving two free sulfhydryl groups.
Based on the empirical data obtained by the inventor and disclosed herein, a cyclic peptide of the invention may have the sequence of SEQ ID NO: 1, or it may be a derivative of that sequence in which the second amino acid is methionine (SEQ ID NO: 17), or in which the 5th amino acid is proline (SEQ ID NO: 5), or in which the 6th amino acid is asparagine (SEQ ID NO: 9), or in which the 7th amino acid is leucine (SEQ ID NO: 3), or in which the 8th amino acid is arginine (SEQ ID NO: 2), or any combination of these substitutions, or even conservative variants of any of these substitutions.
Another aspect of the present invention includes cyclic peptides inhibitors of LFA-1/ICAM-1 interaction comprising the heptapeptide sequences which include LLRMRSI (SEQ ID NO: 49), LLRMRSA (SEQ ID NO: 50), LIRMRSA (SEQ ID NO: 51), LLKMRSA (SEQ ID NO: 52), LLRMKSA (SEQ ID NO: 53), ILRMRSA (SEQ ID NO: 54) or LLRMRSV (SEQ ID NO: 55). Peptides that comprise the heptapeptide sequences of the present invention may be a peptide of 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 amino acids in length, wherein additional amino acids may be any L-series or any D-series amino acid.
In preferred embodiments of the invention, the peptides or peptide mimetics of the invention exhibit an inhibition constant (IC50) for the binding interaction of LFA-1/ICAM-1 of from about 10 xcexcM to about 800 xcexcM for cell aggregation or from about 10 to about 250 nM for monovalent ICAM-1 binding. The term xe2x80x9cIC50xe2x80x9d is well known in the art, and means the half maximal inhibitory concentration, or concentration at which aggregation is inhibited by 50%.
Any of the compositions described herein may be formulated for pharmacological or therapeutic administration either to a mammal, or more preferably to a human. As such, the compositions may be contained in a pharmaceutically acceptable carrier. The preferred mode of administration of a peptide active agent is by injection, either intravenous, intra-arterial, intramuscular or subcutaneous. Other routes of administration may also be possible and would be included within the scope of the present disclosure.
The compositions may be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be suitably fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
As used herein, xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
A peptide can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
It is an aspect of the present disclosure that the disclosed compositions may be used in adjunct therapy in standard treatments for diseases such as hematopoietic neoplasms. The present invention may be described therefore, in certain embodiments as a method of preventing retinoic acid syndrome in a subject receiving all-trans retinoic acid, comprising administering to the subject an effective amount of a composition comprising a cyclic peptide, wherein the peptide comprises a heptapeptide sequence including, but not limited to, LLRMRSI (SEQ ID NO: 49), LLRMRSA (SEQ ID NO: 50), LIRMRSA (SEQ ID NO: 51), LLKMRSA (SEQ ID NO: 52), LLRMKSA (SEQ ID NO: 53), ILRMRSA (SEQ ID NO: 54) or LLRMRSV (SEQ ID NO: 55). In some embodiments the cyclic peptide has the amino acid sequence CLLRMRSIC (SEQ ID NO: 1) or CLLRMRSAC (SEQ ID NO: 40), or a conservative variant thereof, such as CILRMRSAC (SEQ ID NO: 43), CLIRMRSAC (SEQ ID NO: 44), CLLKMRSAC (SEQ ID NO: 45), CLLRMKSAC (SEQ ID NO: 46), or CLLRMRSVC (SEQ ID NO: 48).
An embodiment of the invention may also be described as a method for inhibiting growth of leukemia cells comprising preventing an LFA-1/ICAM-1 interaction between the leukemia cells and support cells such as bone marrow stromal cells, wherein the method comprises contacting the leukemia cells with a cyclic peptide inhibitor of the LFA-1/ICAM-1 interaction, and further wherein the amino acid sequence of the cyclic peptide inhibitor is not a fragment of the amino acid sequence of LFA-1 or ICAM-1. In the practice of this method, the leukemia cells are preferably in a leukemia patient and contacting comprises administering the cyclic peptide to the patient.
An embodiment of the invention is also a therapeutic package for dispensing to, or for use in dispensing to, a mammal or human being treated for a hematopoietic neoplastic disease, myocardial infarction, radiation injury, asthma, rheumatoid arthritis, or lymphoma metastasis, wherein the package contains in a unit dose, an amount of a cyclic peptide comprising a heptapeptide sequence including, but not limited to, LLRMRSI (SEQ ID NO: 49), LLRMRSA (SEQ ID NO: 50), LIRMRSA (SEQ ID NO: 51), LLKMRSA (SEQ ID NO: 52), LLRMKSA (SEQ ID NO: 53), ILRMRSA (SEQ ID NO: 54) or LLRMRSV (SEQ ID NO: 55), effective to inhibit an LFA-1/ICAM-1 interaction in a subject when administered periodically. In some embodiments the cyclic peptide has the amino acid sequence CLLRMRSIC (SEQ ID NO: 1) or CLLRMRSAC (SEQ ID NO: 40), or a conservative variant thereof, such as CILRMRSAC (SEQ ID NO: 43), CLIRMRSAC (SEQ ID NO: 44), CLLKMRSAC (SEQ ID NO: 45), CLLRMKSAC (SEQ ID NO: 46), or CLLRMRSVC (SEQ ID NO: 48). In certain embodiments a unit dose is from about 10 to about 500 xcexcg/Kg, or is from about 50 to about 250 xcexcg/Kg, or from about 120 to about 150 xcexcg/Kg. The unit dose may be an initial bolus dose which may be followed by a continuous infusion of about 5 to about 250 xcexcg/Kg/min, or from about 40 to about 60 xcexcg/Kg/min, or may be about 50 xcexcg/Kg/min. Alternatively, unit doses may be repeated daily, and administered multiple times per day. Dosage regimens are not limited to those exemplified, and the invention encompasses any dosage regimen that delivers an therapeutically effective dose. An example of clinical administration of a peptidomimetic inhibiting an integrin functions is documented by Ohman et al. (1995).
An embodiment of the present disclosure is a method of inhibiting emigration of leukocytes from blood into tissue in a subject comprising administering to the subject an amount of a cyclic peptide comprising a heptapeptide sequence including, but not limited to, LLRMRSI (SEQ ID NO: 49), LLRMRSA (SEQ ID NO: 50), LIRMRSA (SEQ ID NO: 51), LLKMRSA (SEQ ID NO 52), LLRMKSA (SEQ ID NO: 53), ILRMRSA (SEQ ID: 54) or LLRMRSV (SEQ ID NO: 55), effective to inhibit an LFA-1/ICAM-1 interaction in the subject. In some embodiments the cyclic peptide has the amino acid sequence, CLLRMRSIC (SEQ ID NO: 1) or CLLRMRSAC (SEQ ID NO: 40), or a conservative variant thereof, such as CILRMRSAC (SEQ ID NO: 43), CLIRMRSAC (SEQ ID NO: 44), CLLKMRSAC (SEQ ID NO 45), CLLRMKSAC (SEQ ID NO:46), or CLLRMRSVC (SEQ ID NO: 48). In preferred embodiments the subject is susceptible to the development of or is suffering from a hematopoietic neoplastic disease, myocardial infarction, radiation injury, asthma, rheumatoid arthritis, or lymphoma metastasis.
A further embodiment of the invention is a method comprising a competitive binding assay for screening the ability of candidate compounds to bind to ICAM-1. The method comprises assessing the displacement of candidate compounds by cyclic petide inhibitors of LFA-1/ICAM-1 binding interaction. The cyclic peptide may comprise a heptapeptide sequence including, but not limited to, LLRMRSI (SEQ ID NO: 49), LLRMRSA (SEQ ID NO: 50), LIRMRSA (SEQ ID NO: 51), LLKMRSA (SEQ ID NO 52), LLRMKSA (SEQ ID NO: 53), ILRMRSA (SEQ ID NO: 54) or LLRMRSV (SEQ ID NO: 55). In some embodiments the cyclic peptide has the amino acid sequence, CLLRMRSIC (SEQ ID NO: 1) or CLLRMRSAC (SEQ ID NO: 40), or a conservative variant thereof, such as CILRMRSAC (SEQ ID NO: 43), CLIRMRSAC (SEQ ID NO: 44), CLLKMRSAC (SEQ ID NO: 45), CLLRMKSAC (SEQ ID NO: 46), or CLLRMRSVC (SEQ ID NO: 48).
Other embodiments of the present disclosure include methods of therapy relating to tissue allografts such as renal, heart and thryoid allografts, bone marrow transplants, diabetes, rheumatoid arthritis, psoriasis, T-cell mediated sensitization reactions such as contact sensitization, and other T-cell mediated disorders. Such methods comprise comprising administering to the subject an amount of a cyclic peptide comprising a heptapeptide sequence including, but not limited to, LLRMRSI (SEQ ID NO: 49), LLRMRSA (SEQ ID NO: 50), LIRNIRSA (SEQ ID NO: 51), LLKMRSA (SEQ ID NO: 52), LLRMKSA (SEQ ID NO:53), ILRMRSA (SEQ ID NO: 54) or LLRMRSV (SEQ ID NO: 55), effective to inhibit an LFA-1/ICAM-1 interaction in the subject. In some embodiments the cyclic peptide has the amino acid sequence, CLLRMRSIC (SEQ ID NO: 1) or CLLRMRSAC (SEQ ID NO: 40), or a conservative variant thereof, such as CILRMRSAC (SEQ ID NO: 43), CLIRMRSAC (SEQ ID NO: 44), CLLKMRSAC (SEQ ID NO: 45), CLLRMKSAC (SEQ ID NO: 46), or CLLRMRSVC (SEQ ID NO: 48).